1. Field of the Invention
This invention relates to specific DNA sequences which are capable of expressing foreign proteins and their metabolites in dicotyledonous plants and cell culture. Specifically, the present invention concerns the incorporation into a vector and expressing of the SBAmyB or SBAmyC gene of the sugar beet and recovering the product of the expressed gene.
2. Description of Related Art
The incorporation of the sugar beet genes SBAmyB or SBAmyC for α-amylase into other plants and seeds (particularly dicots) for expression has not been previously reported in the prior art.
The synthesis of α-amylase and levels of their mRNA are greatly induced under sucrose starvation. An increase of α-amylase synthesis is assumed to accelerate hydrolysis of cellular starch as an energy source when exogenous carbon source is depleted. Under normal growth condition with an adequate supply of sugars in the medium, the expression of α-amylase genes is subject to metabolite repression. It was observed that α-amylases synthesized by the cultured rice cells are secreted into the culture medium and can account for about 15–20% of the total proteins present in the medium during periods of sugar depletion.
It would therefore be advantageous to develop a gene expression system in plant cell culture by constructing a vector expressible in plant host cells utilizing the promoter and the signal peptide sequences of an α-amylase gene. Any foreign gene can be linked downstream of said promoter and signal peptide encoding sequences. This construct would then be used to transform a compatible plant host cell.
Theoretically, the α-amylase promoter would control the expression of foreign genes in said plant cells and the secretion of the proteins into the medium. Such an expression system therefore has a high potential to express and/or secrete large quantities of any important protein into the medium, greatly facilitating purification of the expressed protein.
To aid in the procedure of screening and/or to enhance further the expression efficiency of the gene expression system constructed above, said gene expression system may further comprise a suitable marker gene, a reporter gene, an antibiotic-resistance gene and/or an enhancer gene, all of which can be those well known by one of ordinary skill in the relevant art (see Maniatis, T., et al, “Molecular Cloning: A Laboratory Manual,” pressed by Cold Spring Harbor Laboratory, 2nd ed., 1989).
Some references of interest in this field include the following:
M. T. Chan et al. in The Journal of Biological Chemistry, vol. 269 (#26), pp. 17635–17641 discloses a novel gene expression system for plant cells based on the induction of α-amylase promoter by carbohydrate starvation. Specifically the 5′-regulatory region and putative signal sequence of a rice α-amylase gene. α-BSAmyA was fused into bacterial gene and introduced into rice, tobacco and potato systems.
R. L. Rodriguez, U.S. Pat. No. 5,888,789 discloses a process for protein production in plants.
R. L. Rodriguez in U.S. Pat. No. 6,048,973 discloses sugar-regulatory sequences in alpha amylase genes.
S.-M. Yu et al. in U.S. Pat. No. 5,460,952 describe gene expression comprising the promoter region of the alpha-amylase alpha-anylase genes. Specifically, a method is described for expression of Amy1 6, Amy1 7, Amy1 8 and Amy1 10 genes from rice.
B. Li et al., (1992) Plant Physiology, vol. 98, pp. 1277–1284 disclose research concerning the characterization and subcellular localization of debranching enzyme and endoamylase from the leaves of sugar beet.
B. R. Thomas et al, (1994) Plant Physiol., vol. 106, pp. 1235–1239 teach metabolite signals which regulate gene expression and source/sink relations in cereal seedlings. During the seedling elongation stage, the Amy1A gene is expressed at a high level in the rice aleurone. The Amy3B, Amy3C and Amy3E genes are expressed at moderate levels. There is little or no expression of Amy 1B, Amy 1C, Amy 2A, or Amy 3A in the developing rice seedling.
D. Yamanouchi et al (1990), Nucleic Acids Res., vol. 18 (#14), 4250, disclose the nucleic acid sequence of cDNA for α-amylase from cotyledon of germinating vigna mungo seeds.
D. M. Lawrence et al (1990) Physiologia Plantarium, vol. 78, pp. 421–429 disclose the mobilization of storage reserves during germination and early seedling growth of the sugar beet.
J. W. Kim et al (1997) J. Plant Res., vol. 110, pp. 357–361 disclose the expression of α-amylase in cultured callus of French bean.
H. Takeuchi et al (1993) Plant Physiol. (Plant Gene Register), vol. 103, p. 1459 disclose the nucleotide sequence of the α-amylase gene from vigna mungo. 
All patents, patent applications, articles, reference standards cited herein are incorporated by reference in their entirety.
The problem remains to successfully incorporate the genes of the sugar beet into other plants and seeds (particularly dicots) for expression to produce improved growth and biological properties. The present invention provides a novel method.